The analysis of gene function has increasingly come to require the production of subtle, tissue-specific, and conditional mutations in animals and plants. Although there are a number of methods for engineering subtle mutations in embryonic stem (ES) cells (Hasty et al. (1991) Nature 350:243-246, Askew et al. (1993) Mol Cell Bio) 13:4115-4124), the use of site-specific recombinases to remove the selectable marker that permits isolation of homologously recombined ES cell clones has become increasingly prevalent (Kitamoto et al. (1996) Biochem Diophys Res Commun 222:742-747, Fiering et al. (1993) Proc Natl Acad Sci USA 90:8469-8473, Schwenk et al. (1995) Nucleic Acids Res 23:5080-5081; Gu et al. (1993) Cell 13:1155-1164; Sailer et al. (1996) Taniguchi Symposia on Brain Sciences, eds. Hakanishi et al. (Japan Scientific Press), pp. 89-98).
Site-specific recombinases represent the best method for creating tissue-specific and conditional mutations in animals and plants being employed first to remove the selectable marker to create a functionally wild-type allele, and then to inactivate the allele mosaically in animals and plants by removing some essential component in a tissue-specific or conditional manner (Ciu et al. (1994) Science 265:103-106; Kuhn et al. (1995) Science 269:1427-1429). Current protocols for using excessive site-specific recombination to remove selectable markers include transiently transfecting ES cell clones with a recombinase expression vector (Gu et al., (1993) Cell 73:1155-1164), microinjecting fertilized oocytes containing the recombinant allele with a recombinase expression vector (Kitamoto et al. (1996) Biochem Biophys Res Commun 222:742-747; Araki et al. (1995) Proc Natl Acad Sci USA 92:160-164), or breeding animals and plants containing the recombinant allele to animals and plants, respectively, containing a recombinase transgene (Schwenk et al. (1995) Nucleic Acids Res 23:5080-5081; Lewandoski et al. (1997) Curr Biol 7:148-151). Each of these approaches requires an investment of some combination of time, resources, and expertise over that required to generate animals and plants with homologously recombined alleles. The most commonly employed method, the secondary transfection of homologously recombined ES cell clones with a recombinase expression vector, additionally requires extended culture time that may decrease their potential to enter the germline.
In principle, marker excision would be substantially simplified through the use of ES cells containing recombinase nucleic acid constructs that were expressed in the germline, but not to an appreciable extent in the ES cells themselves or somatic tissues of animals and plants. The lack of ES cell expression would mean that targeting vectors containing selectable markers flanked by recombinase target sites could be used to isolate homologous recombinants without fear that the marker would be excised during culture. Robust recombinase expression in gametes would mean that the marker would be excised in at least some of the progeny of ES cell chimeras. Only a single step would be required to isolate subtle mutations and, if two different recombinase systems were employed, conditional and tissue-specific alleles could be produced with similar improvements in efficiency. A germline-specific recombinase nucleic acid construct could also be used to deliver recombined target nucleic acid constructs to the early embryo (Lewandoski et al. (11997) Curr Biol 7:148-151), so long as the recombined target was not detrimental to the terminal stages of spermatogenesis.
Previous reports have shown that expression of nucleic acid constructs containing the proximal promoter of the mouse protamine 1 (mP1) locus is restricted to haploid spermatids in mature mice (Peschon et al. (1987) Proc Natl Acad Sci USA 84:5316-5319; Behringer et al. (1988) Proc Natl Acad Sci USA 85: 2648-2652) although low levels of ectopic expression may occur in some mature tissues (Behringer et al. (1988) Proc Natl Acad Sci USA 85:2648-2652). Inclusion of the mP1 promoter does not guarantee expression in the male germline, however, for although nucleic acid constructs containing the mP1 promoter and the SV40 T-antigen coding sequence were transcribed, the message was not translated at detectable levels in spermatids (Behringer et al. (1988) Proc Natl Acad Sci USA 85: 2648-2652).
Accordingly, there is a need in the art for methods to modulate expression of recombined target nucleic acid sequences in the early embryo. In addition, there is a need in the art for tissue-specific and conditional recombinatory tools to create transgenic animals and plants. These and other needs in the art are addressed by the present invention.